Bacterial secretome for use in the treatment of skin lesions
a technology of skin lesions and secretomes, applied in the field of skin lesions treatment, can solve the problems of unsightly, mostly permanent visible marks, and cosmetic problems of healing
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example 1
F BACTERIUM LMB64
[0117]By way of non-limiting example, preferred culture media contain ammonium chloride, magnesium sulphate and yeast extract. It should also be noted that, as arises from application WO2012 / 085182, among others, other similar media may be used and must thus be regarded as forming an integral part of the present description. Any adaptation by persons skilled in the art must also be regarded as part of the invention.
[0118]An exemplary culture process is described below. It should be recalled here that this example is for illustrative purposes only and must in no way be regarded as limiting.
[0119]Strain LMB64 is grown in three steps, namely a first inoculum, a pre-culture (or pre-fermentation) in batch mode and finally a culture but in fed-batch mode (addition of glucose).
[0120]Inoculum:
[0121]A tube of WCB LMB64 is used to inoculate an Erlenmeyer flask containing 1000 mL of sterile medium. The Erlenmeyer flask is then placed in the incubator shaker and shaken. When th...
example 2
N OF THE SECRETOME
[0136]The example below is given by way of illustration of a preferred embodiment, but must not be regarded as limiting.
[0137]The secretome is obtained, generally, after centrifugation of the result of the culture step in order to remove cells, surface proteins and proteins located in the periplasmic space of the bacterium. This centrifugation step is followed by an addition of a Tris-arginine basic buffer to the supernatant. A last step of filtration of the supernatant is also possible. Any adaptation by persons skilled in the art must also be regarded as part of the invention.
[0138]Centrifugation:
[0139]The transfer line from the fermenter to the centrifuge is sterilized. The fermentation must is then separated by continuous centrifugation on a centrifuge. Centrifugation is performed at 150 L / h (±30 L / h) with a bowl rotation speed of 10900±1000 rpm. The supernatant is collected in a container fitted with a disposable pouch. The weight of the supernatant is measure...
example 3
THE SECRETOME ON PROLIFERATION OF NORMAL HUMAN FIBROBLASTS
[0146]The technique used is that of incorporation of a nucleotide, 5-bromo-2′-deoxyuridine (BrdU), a thymidine analogue, in the DNA of S-phase cells, at 37° C. This technique allows quantification of the cells whose progression in the cell cycle is characteristic of a proliferative cell (S or DNA synthesis phase).
[0147]Fibroblasts are seeded in a 96-well plate and then incubated for 72 h in the presence of the compounds to be tested, EGF as positive control and 0.4, 0.6 and 1% secretome. BrdU incorporation is performed during the last 24 h and is quantified using the BrdU ELISA kit (item no. 11647229001, Roche Diagnostics).
[0148]The % of the control is calculated according to the formula:
(Value / mean of the control)×100
[0149]Stimulation in % is calculated according to the formula:
Mean Stimulation in %=(Mean(Value / mean of the control)×100)−100
[0150]SEM=standard dev / √n
[0151]Statistical analyses are performed using Student's t-te...
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